The laser-scanning microscope (LSM) represents a modern tool for observing smaller structures [1]. As with the traditional microscope, the object is initially imaged via the objective and tube lenses onto the intermediate image level. In a second imaging step of the LSM the confocal principle is realized on the excitation and detection sides. On the excitation side, the launching occurs by means of a punctiform fiber output via a collimator [2], or directly into the infinite space in front of the scanning objective lens, which is focused onto the intermediate image level of the traditional microscope alignment. For the detection the spectrally staggered fluorescent radiation originating from the objective is used, which falls onto a pinhole for detection via the scanning objective and pinhole optics after the intermediate image level. Excitation and detection channels are separated by means of color separators. With the aid of scanning mirrors, which are arranged in the infinite space between the scan objective and collimator/pinhole optics adjacent of the image of the objective pupil, each image field point can be illuminated and detected. The image is then electronically composed of a detector signal and of the information on the scanner positions. The radiation formation occurs in the objective pupil (FIG. 1).
Due to the fact that optics are often used that correspond to the VIS requirements of traditional light microscopy up to the intermediate image, focal differences occur for the spectral ranges (UV, IR) newly to be developed in dependency of the correction made by the objective and tube lenses. Any systematically occurring parts of lens distortions or aberrations can be compensated in the scanning objective lens. Currently, any chromatic distortions beyond this are compensated by a spectral breakdown of the launching and pinhole optics into channels of varying optical path lengths.
But substantial mechanical adjustment paths often occur even within one channel. This is caused by the mostly strong wavelength dependency of the chromatic distortion within the UV and IR ranges. On the other hand, systems with a high lateral imaging scale (˜100) show a sensitive behavior with regard to the transformation of axial position changes from the object into the image space.
One solution is the staggering of the entire collimator group that is positioned conjugated to the pinhole level, which can supply a sufficiently large logarithm for the defocusing up to a certain degree [2]. It differs from solutions that are aimed at varying the illumination diameter by means of variooptics [3].
Literature:
[1] Wilson, Confocal Microscopy, Academy Press
[2] Published Application DE 19702753A1
[3] DE 19654211 A1,
DE 19901219 A1